Hepatitis C virus (HCV) depends on host lipid metabolism for its lifecycle, including entry, replication and assembly. The liver-abundant microRNA miR-122 is a host factor required for viral replication. This pro-viral mechanism is in part mediated by direct binding to the 5' UTR of the viral genome. However, direct binding does not completely account for the observed effects on viral replication. miR-122 has also been demonstrated to regulate cholesterol metabolism through unknown mechanisms. Using an in vitro model examining HCV viral replication, we confirmed that cholesterol biosynthetic intermediates did not rescue miR-122- suppressed HCV replication. However, LDL effectively restored replication. Furthermore, we demonstrated that apolipoprotein B100 (apoB100) itself rescued replication from miR-122 antagonism, and alone robustly promotes viral replication. In turn, RNAi-mediated knockdown of apoB100 attenuated the proviral effect of miR122, and cell lines with biallelic deletion of the APOB gene support only low levels of viral replication with HCV, and miR-122 overexpression fails to enhance replication, compared to a 2.5-fold increase observed in the wild type cells. These data indicate that miR122 exerts a pro-viral effect that is mediated in part by apoB100, and that apoB100 is a necessary host factor to support HCV replication. We have additionally demonstrated that miR-122 antagonism leads to a significant reduction in apoB100 expression at the protein, but not mRNA level. There is not presently a known mechanistic link between miR122 function and lipoprotein expression. In this proposal, we will (1) determine the mechanism(s) by which apoB100 supports HCV replication; and (2) characterize the mechanisms by which miR-122 regulates expression of apoB100. We will clarify the regulatory pathways linking miR-122 and apoB100 by assessing the impact of miR-122 on proteins known to be critical for post-translational regulation of ApoB100. We will also examine whether miR-122 indirectly regulates cellular cholesterol metabolism through its impact on other microRNAs, and microRNA-33, in particular, given its known involvement in cholesterol synthesis. Taken together, these studies will serve to clarify the relationship between microRNA-122, HCV, and lipid metabolism and provide insights into the pathogenesis of chronic hepatitis and its related derangements, including steatosis.